The rapid genetic changes of influenza viruses continue to cause great difficulties with current strain- specific influenza vaccines in providing adequate control for influenza epidemics and effective countermeasures against pandemics. Thus, a universal vaccine that targets conserved antigen domains and provides protection against all influenza virus strains is urgently needed. The M2e and fusion peptide (FP) are the two most highly conserved antigen domains found in influenza virus surface proteins. The M2e (24 amino acids) is conserved among all influenza A viruses and the FP (14 amino acids) is conserved among both influenza A and B viruses. Current development efforts for a universal vaccine have primarily focused on single-domain constructs based on M2e. As the conserved antigen domains consist of just short peptides, an effective multivalent protein carrier system is critical in enhancing vaccine effectiveness and product feasibility with respect to manufacturing, storage, distribution, and use. KJ Biosciences aims to meet this urgent need by developing a novel dual-domain universal influenza vaccine incorporating both M2e and FP antigen domains that will provide protection against all influenza A and B viruses and can therefore be used to control influenza epidemics as well as pandemics. It will be produced by dual-domain fusion with a unique nanoparticle protein carrier that has both N- and C-termini exposed on the surface for antigen attachment. The vaccine product can also be stabilized at room or higher temperatures based on the unique properties of the carrier protein. A thermostable vaccine will significantly reduce the logistical requirements for storage, distribution, and use of the vaccine product, which is especially important when facing a pandemic. The goal of this proposal is to continue to develop this dual-domain universal influenza vaccine based on the promising results from preliminary studies. The proposed studies have two specific aims. Specific aim 1: Generation and characterization of the fusion proteins. Dual-domain fusion proteins with M2e and FP antigen domains arranged in different positions will be generated with the nanoparticle carrier protein by recombinant DNA techniques. They will be tested to demonstrate nanoparticle formation, reaction with antibodies specific to the antigen domains, and stability at room or higher temperatures. Specific aim 2: Immunogenicity and protection. Selected dual-domain fusion protein vaccine candidate (s) will be tested in animal models to demonstrate that the vaccine candidate can induce specific antibodies and protect animals against challenge with live influenza viruses. Completion of these two specific aims will form the basis for further preclinical immunogenicity evaluation and process development to produce the candidate vaccine for toxicology and phase I clinical studies. Successful development of this vaccine will afford an effective measure for controlling influenza epidemics and also greatly enhance our ability to respond to future pandemics.